Microtubule depolymerization can drive poleward chromosome motion in fission yeast

Abstract

Prometaphase kinetochores interact with spindle microtubules (MTs) to establish chromosome bi-orientation. Before becoming bi-oriented, chromosomes frequently exhibit poleward movements (P-movements), which are commonly attributed to minus end-directed, MT-dependent motors. In fission yeast there are three such motors: dynein and two kinesin-14s, Pkl1p and Klp2p. None of these enzymes is essential for viability, and even the triple deletion grows well. This might be due to the fact that yeasts kinetochores are normally juxtapolar at mitosis onset, removing the need for poleward chromosome movement during prometaphase. Anaphase P-movement might also be dispensable in a spindle that elongates significantly. To test this supposition, we have analyzed kinetochore dynamics in cells whose kinetochore-pole connections have been dispersed. In cells recovering from this condition, the maximum rate of poleward kinetochore movement was unaffected by the deletion of any or all of these motors, strongly suggesting that other factors, like MT depolymerization, can cause such movements in vivo. However, Klp2p, which localizes to kinetochores, contributed to the effectiveness of P-movement by promoting the shortening of kinetochore fibers.

Figure 1

‘Lost' kinetochores are successfully retrieved and bi-orientated. (A) At 18°C cells arrested with condensed chromosomes that remained clustered together (‘condensed chromosomes'). In many such cells at least one kinetochore lost its connection with SPBs (‘lost kinetochores'), and some had separated SPBs. The error bars are standard errors of the mean (s.e.m.) with 95% confidence everywhere in the manuscript unless stated otherwise. (B–D) Progress though mitosis was monitored by fixing cultures sampled at the indicated times after the shift to 32°C and immunostaining. Control—black circles, pkl1Δ—white circles, klp2Δ—white squares, dhc1Δ—black squares. Numbers in (B) and (D) are normalized to the percent at time 0. (B) Percent of cells in which at least one of the kinetochores is neither close to the SPB(s), nor is it positioned on the line between the separated SPBs (see E (a–d)). (C) The percent of cells in which the SPBs are separated and all kinetochores are found on the line between the SPBs, that is, either mono- or bi-oriented (see E, e–g). (D) Percent of cells with condensed chromosomes that are clustered together. The deviations between control and either klp2Δ or pkl1Δ cells are statistically significant (P<0.05), but dhc1Δ and control cells cannot be distinguished. (E) Representative images from synchronized cultures fixed 3–10 min after the shift. Bar: 2 μm. (F) Selected images of an nda3 cell as it recovers from a cold-block, showing kinetochore retrieval and bi-orientation.

Figure 2

Kinetics and structure of initial chromosome–MT interaction. (A, B) Trajectories of kinetochore movements prior to (red line) and after (blue line) the temperature shift to 32°C in two control cells. The images show movements in the plane that contained the kinetochore (red X's) and both spindle poles (see Materials and methods). Blue arrows point to the onset of the final movements that brought these kinetochores to the pole. Purple arrows point to pauses. Final positions for the kinetochores and poles are shown as red and green circles, respectively. The grid squares are 0.5 μm. (C) A slice from a tomogram, built from nda3 cell frozen during early stage of spindle formation. (D–F) Reconstructions of three spindles (frozen 3–7 min after the shift to 32°C), one of which is shown in (C). MTs nucleated from the different poles are colored green and red, spindle poles are yellow, and the nuclear envelope is gray. Minus ends are shown with orange dots, plus ends with purple dots; ends that lie between the sections are not marked. Occasionally, there were MTs that continued beyond the reconstructed sections; their most distal (plus) ends were marked with green dots. Bar: 0.1 μm.

Figure 3

Movements during chromosome retrieval in live cells. (A, B) Example traces of the kinetochore-to-pole distances versus time after the shift to 32°C (see also Supplementary Figure 6). (C) Comparison of the slopes of final kinetochore approaches for the klp2Δ cell 1 (Supplementary Video 5), klp2Δdhc1Δ cell (Supplementary Video 12), one control cell and 4 pkl1Δ klp2Δ dhc1Δ cells.

Figure 4

Klp2p facilitates kinetochore retrieval. (A) 2D trajectory of kinetochore retrieval in cell 3. The best fitting circular arc (magenta) for frames 1–50 (Supplementary Video 7); a best linear fit (blue) for frames 51–96. Tomographic slice (B) and 3D model (C) of nda3klp2Δ cell delayed in prometaphase (frozen 15 min after the shift to 32°C). Arrow in (B) points to one of the MTs in a two-MTs bundle growing from the right pole. Bar: 100 nm. (D) Average numbers of MTs per spindle pole for control nda3 cells (4 poles), nda3dhc1Δ (10), nda3pkl1Δ (6) and delayed nda3klp2Δ cells (6). (E) ET slices of the MTs plus ends. The first five images were collected from a control nda3 cell; all the rest are from six klp2Δ cells. There is a great variability in the appearance of the plus ends in both normal and mutant cells, which makes it difficult to quantify differences. Usually, the plus MT ends are splayed open (upper row), but in klp2Δ cells they frequently contain occlusions (red arrows). The images in middle row may represent intermediates that lead to ‘scarred' MTs with altered polymerization behavior; the bottom row suggests a possible pathway for the rescue of rapid shortening at an MT abnormality, followed by renewed MT polymerization. Bar: 100 nm. (F) Percent of MTs plus ends that show darker ‘bars' inside the MT lumen. Number of ends examined: control 68, klp2Δ 239. (G) Selected live images (30 s apart) from an nda3 klp2Δ dhc1Δ cell undergoing anaphase A. Bar: 2 μm.

Figure 5

P-movement and bi-orientation of chromosomes in S. pombe. As normal mitosis begins, the SPBs (green ovals) are close to the kinetochores (red circles; only one chromosome (blue) is shown); this situation may explain why all three minus-end directed motors are not required for mitotic progression. In the absence of Klp2p, or during transient MT depolymerization, the kinetochores can move some distance away from the poles. Their subsequent movement to the pole is driven by MT depolymerization, facilitated by Klp2.